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Thursday, February 16, 2017

The Mysterious 98%: Scientists Look to Shine Light on Our Dark Genome

UCSF: After the 2003 completion of the Human Genome Project – which sequenced all 3 billion “letters,” or
base pairs, in the human genome – many thought that our DNA would
become an open book. But a perplexing problem quickly emerged: although
scientists could transcribe the book, they could only interpret a small
percentage of it. The mysterious majority – as much as 98 percent – of our DNA do not
code for proteins. Much of this “dark matter genome” is thought to be
nonfunctional evolutionary leftovers that are just along for the ride.
However, hidden among this noncoding DNA are many crucial regulatory
elements that control the activity of thousands of genes. What is more,
these elements play a major role in diseases such as cancer, heart
disease, and autism, and they could hold the key to possible cures.
As part of a major ongoing effort to fully map and annotate the
functional sequences of the human genome, including this silent
majority, the National Institutes of Health (NIH) on Feb. 2, 2017, announced new grant funding for a nationwide project
to set up five “characterization centers,” including two at UC San
Francisco, to study how these regulatory elements influence gene
expression and, consequently, cell behavior.
The project’s aim is for scientists to use the latest technology,
such as genome editing, to gain insights into human biology that could
one day lead to treatments for complex genetic diseases.

Importance of Genomic Grammar

After the shortfalls of the Human Genome Project became clear, the Encyclopedia of DNA Elements (ENCODE) Project
was launched in September 2003 by the National Human Genome Research
Institute (NHGRI). The goal of ENCODE is to find all the functional
regions of the human genome, whether they form genes or not.

The Human Genome Project mapped the
letters of the human genome, but it didn’t tell us anything about the
grammar: where the punctuation is, where the starts and ends are.

Elise Feingold, PhD

NIH Program Director

“The Human Genome Project mapped the letters of the human genome, but
it didn’t tell us anything about the grammar: where the punctuation is,
where the starts and ends are,” said NIH Program Director Elise
Feingold, PhD. “That’s what ENCODE is trying to do.”
The initiative revealed that millions of these noncoding letter
sequences perform essential regulatory actions, like turning genes on or
off in different types of cells. However, while scientists have
established that these regulatory sequences have important functions,
they do not know what function each sequence performs, nor do they know
which gene each one affects. That is because the sequences are often
located far from their target genes – in some cases millions of letters
away. What’s more, many of the sequences have different effects in
different types of cells.
The new grants from NHGRI will allow the five new centers to work to
define the functions and gene targets of these regulatory sequences. At
UCSF, two of the centers will be based in the labs of Nadav Ahituv, PhD, and Yin Shen,
PhD. The other three characterization centers will be housed at
Stanford University, Cornell University, and the Lawrence Berkeley
National Laboratory. Additional centers will continue to focus on
mapping, computational analysis, data analysis and data coordination.

Cellular Barcodes Reveal Regulatory Function

New technology has made identifying the function and targets of
regulatory sequences much easier. Scientists can now manipulate cells to
obtain more information about their DNA, and, thanks to high-throughput
screening, they can do so in large batches, testing thousands of
sequences in one experiment instead of one by one.
“It used to be extremely difficult to test for function in the
noncoding part of the genome,” said Ahituv, a professor in the
Department of Bioengineering and Therapeutic Sciences. “With a gene,
it’s easier to assess the effect because there is a change in the
corresponding protein. But with regulatory sequences, you don’t know
what a change in DNA can lead to, so it’s hard to predict the functional
output.”
Ahituv and Shen are both using innovative techniques to study
enhancers, which play a fundamental role in gene expression. Every cell
in the human body contains the same DNA. What determines whether a cell
is a skin cell or a brain cell or a heart cell is which genes are turned
on and off. Enhancers are the secret switches that turn on cell-type
specific genes.Nadav Ahituv (right), PhD, is using new technology to test for enhancers among 100,000 regulatory sequences in DNA. Photo by Susan MerrellDuring
a previous phase of ENCODE, Ahituv and collaborator Jay Shendure, PhD,
at the University of Washington, developed a technique called
lentivirus-based massive parallel reporter assay to identify enhancers.
With the new grant, they will use this technology to test for enhancers
among 100,000 regulatory sequences previously identified by ENCODE.
Their approach pairs each regulatory sequence with a unique DNA
barcode of 15 randomly generated letters. A reporter gene is stuck in
between the sequence and the barcode, and the whole package is inserted
into a cell. If the regulatory sequence is an enhancer, the reporter
gene will turn on and activate the barcode. The DNA barcode will then
code for RNA in the cell.
Once the researchers see that the reporter gene is turned on, they
can easily sequence the RNA in the cell to see which barcode is
activated. They then match the barcode back to its corresponding
regulatory sequence, which the scientists now know is an enhancer.
“With previous enhancer assays, you had to test each sequence one by
one,” Ahituv explained. “With our approach, we can clone thousands of
sequences along with thousands of barcodes and test them all at once.”

Deleting Sequences to Understand Their Role

Shen, an assistant professor in the Department of Neurology and the
Institute for Human Genetics, is taking a different approach to
characterize the function of regulatory sequences. In collaboration with
her former mentor at the Ludwig Institute for Cancer Research and UC
San Diego, Bing Ren, PhD, she developed a high-throughput CRISPR-Cas9
screening method to test the function of noncoding sequences. Now, Shen
and Ren are using this approach to identify not only which sequences
have regulatory functions, but also which genes they affect.
Shen will use CRISPR to edit tens of thousands of regulatory
sequences in a large pool of cells and track the effects of the edits on
a set of 60 pairs of genes that commonly co-express.Yin
Shen (left), PhD, will use CRISPR to to identify not only which
noncoding sequences of DNA have regulatory functions, but also which
genes they affect. Photo by Susan MerrellFor
this work, each cell will be programmed to reflect two fluorescent
colors – one for each gene – when a pair of genes is turned on. If the
light in a cell goes out, the scientists will know that its target gene
has been affected by one of the CRISPR-based sequence edits. The final
step is to sequence each cell’s DNA to determine which regulatory
sequence edit caused the change in gene expression.
By monitoring the colors of co-expressed genes, Shen will reveal the
complex relationship between numerous functional sequences and multiple
genes, which was beyond the scope of traditional sequencing techniques.
“Until the recent development of CRISPR, it was not possible to
genetically manipulate non-coding sequences in a large scale,” said
Shen. “Now, CRISPR can be scaled up so that we can screen thousands of
regulatory sequences in one experiment. This approach will tell us not
only which sequences are functional in a cell, but also which gene they
regulate.”

Can Dark Matter DNA Treat Disease?

By cataloging the functions of thousands of regulatory sequences,
Shen and Ahituv hope to develop rules about how to predict and interpret
other sequences’ functions. This would not only help illuminate the
rest of the dark matter genome, it could also reveal new treatment
targets for complex genetic diseases.The
causes of common diseases such as diabetes, cancer and autism are not
just a gene that is changed, but the regulatory sequence in human DNA
that regulates that gene, studies have shown.“A
lot of human diseases have been found to be associated with regulatory
sequences,” Ahituv said. “For example, in genome-wide association
studies for common diseases, such as diabetes, cancer and autism, 90
percent of the disease-associated DNA variants are in the noncoding DNA.
So it’s not a gene that’s changed, but what regulates it.”
As the price for sequencing a person’s genome has dropped
significantly, there is talk about using precision medicine to cure many
serious diseases. However, the hurdle of how to interpret mutations in
noncoding DNA remains.
“If we can characterize the function and identify the gene targets of
these regulatory sequences, we can start to reveal how their mutations
contribute to diseases,” Shen said. “Eventually, we may even be able to
treat complex diseases by correcting regulatory mutations.”